I. Technical Field
The present disclosure relates generally to the fields of medicine and oncology genetics. More particularly, it relates to the use of mouse models to screen for pancreatic ductal adenocarcinoma therapeutics, and the use of such therapeutics, alone or in combination, to treat pancreatic ductal adenocarinoma.
II. Related Art
Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer-related deaths but is predicted to become more common due to its association with smoking, diet, obesity, and type 2 diabetes (Pannala et al., 2008; Rahib et al., 2014; Siegel et al., 2015). Three major classifications of pancreatic precancerous lesions are associated with progression to PDA: PanIN (pancreatic intraepithelial neoplasia), IPMN (intraductal papillary mucinous neoplasm) and MCN (mucinous cystic neoplasm) (Distler et al., 2014). Precancerous lesions can be common in the elderly or obese. For example, early PanINs were found in 65% of obese patients, associated with intravisceral fat and pancreatic intralobular fibrosis and fat (Rebours et al., 2015). IPMN are next most commonly associated with PDA. They are found in the pancreatic main and branching ducts. MCN occur predominately in the peripheral pancreas in females.
Recent mathematical predictions attribute spontaneous mutations during cell division as initiators of PDA, making early detection and effective therapy the only two elements determining survival (Tomasetti and Vogelstein, 2015). Unfortunately, PDA symptoms present late, and other than surgical resection, limited progress has been made in developing effective treatment after gemcitabine was introduced as first-line therapy for advanced PDA (Burris et al., 1997). Gemcitabine treatment alone or after resection is marginally effective in prolonging survival. One of the two predominant therapeutic regimens is gemcitabine combined with nab-paclitaxel (Abraxane), which was shown to increase survival to 8.5 months compared with 6.7 months for patients who received gemcitabine alone (Von Hoff et al., 2013). In a follow up study, 12 patients were still alive after 42 months of treatment (Goldstein et al., 2015). In addition to interfering with microtubule function, Abraxane augments gemcitabine efficacy by reducing the level of its metabolizing enzyme, cytidine deaminase (Ibrahim et al., 2002; Frese et al., 2012). However, tumors are often resistant to this combination (Neesse et al., 2014). The other common drug treatment, FOLFIRINOX, consisting of four different chemotherapy agents, is more effective but less well tolerated (Becker et al., 2014; Moorcraft et al., 2014). Therefore, there is a need for a systematic and robust in vivo screen that can accelerate the pace of discovery for improved PDA therapeutics.
PDA initiates as ductal neoplasia, derived from any of three pancreatic adult cell types—ductal progenitor cells, centroacinar cells, or acinar cells that have undergone acinar to ductal metaplasia (ADM) (Bonner-Weir et al., 2004; Rovira et al., 2010; von Figura et al., 2014). In humans, activated Kras and inactivated Cdkn2a are the earliest and most common genetic mutations identified in disease progression (Hezel et al., 2006; Iacobuzio-Donahue et al., 2012). Genetically engineered mouse models (GEMM) based on these mutations have been developed to investigate PDA initiation and propagation. In this report, the inventors use KC (p48Cre;LSL-KrasG12D) and KIC mice (p48Cre;LSL-KrasG12D;Cdkn2af/f). Both lines form tumors because they express activated KrasG12D (KIC also has inactivation of the tumor suppressor Cdkn2a) in all three pancreatic lineages—ducts, acinar and endocrine cells—under control of the p48 (Ptfla) promoter. By contrast, IC mice (p48Cre;Cdkn2af/f) never form tumors. KIC mice are an excellent GEMM for PDA therapeutic screens because neoplasia develops early, between 2 to 3 weeks of age, and large aggressive tumors develop in all mice by 4 weeks of age (Aguirre et al., 2003).
PDA is the most frequent major cancer harboring Ras mutations (e.g., KrasG12D); (Pylayeva-Gupta et al., 2011). Kras mutations are found in over 90% of human PDA (Iacobuzio-Donahue et al., 2012). KrasG12D expression is necessary but not sufficient to initiate neoplasia; GTP binding is required to activate KrasG12D (Huang et al., 2014). Ras guanine nucleotide exchange factors (Ras-GEFs) catalyze GDP dissociation, and subsequent GTP binding to Ras (Jeng et al., 2012). Protein kinase and G-Protein Coupled Receptor (GPCR) signaling can stimulate Ras-GEFs to promote KrasG12D-dependent neoplasia (van Biesen et al., 1995; Kahn, 2014). Regulators of G-protein Signaling (RGS) proteins are GTPase activating proteins (GAPs) for the Gi- and Gq-alpha subunits of heterotrimeric G proteins (Berman et al., 1996). Interestingly, RGS-resistant mutations in Gαq (and Gαs) were found in IPMNs isolated from patients (Wu et al., 2011). RGS proteins are coincidence detectors that can be induced by and integrate multiple inputs to feedback regulate the GPCR arm of the pathway, by virtue of their Gα-GAP activity (Ross and Wilkie, 2000; Huang et al., 2006; Villasenor et al., 2010; Pashkov et al., 2011). The induction of RGS proteins can therefore be monitored to report hyperactivated Ras signaling (Dohlman et al., 1996; Dignard et al., 2008). Because Ras remains an elusive drug target (Stephen et al., 2014), the inventors developed an in vivo screen for PDA therapeutics that is responsive to Kras signaling.
Expression of an Rgs16::GFP bacterial artificial chromosome (BAC) transgene has been shown during embryonic and postnatal pancreas development in pancreatic progenitors, endocrine and duct cells (Villasenor et al., 2010). GFP was expressed in ducts and islet beta cells during neonatal pancreas development but was not detected in euglycemic adult mice. Rgs16::GFP was reactivated, first in ducts, then islet beta cells, under conditions of chronic insulin demand or hyperglycemia in mouse models of type 1 and type 2 diabetes, and during gestation. In humans, Rgs16 expression was observed in ducts of pancreatic cancer patients prior to detectable metastasis (Kim et al., 2010). Chronic stress might induce Rgs16 in progenitor cells within the pancreatic ductal epithelium (Bonner-Weir et al., 2004; Villasenor et al., 2010).